(a) Field of the Invention
The invention relates to a method of regulating the transcription of transgene in genetically-modified organisms. More specifically, the invention relates to the use of expression vectors harboring the coding sequence of a gene of interest under the transcriptional control of promoting sequences for which activity is regulated by the presence of nitrogen. Preferably, these constructs are used in transgenic leguminous plants (for example soybean, alfalfa, clover, birdsfoot trefoil, beans, peas, peanuts) where growth is not impaired by lack of mineral nitrogen, and in which induction of expression could be performed at any given time during development, through the addition of a suitable nitrogen source. In a broader perspective, the invention could be used to induce expression of any given transgene through the addition of any nitrogen source, provided that the organism can be grown adequately in the absence of this nitrogen inducer; as an example within the plant kingdom, duckweed (Lemna minor) can adapt to grow either on nitrate or ammonium as nitrogen source; transgenic duckweed could therefore be grown on nitrate as a sole nitrogen source and expression of the transgene triggered by the addition of ammonium, provided that the cassette contains a promoter from a native gene for which expression is turned on by the addition of ammonium. The invention therefore provides a means of regulating the expression of a transgenic trait in any organism through the addition of various nitrogenous inducer.
(b) Description of Prior Art
Nitrogen is a molecule essential to life. All living organism need nitrogen in order to synthesize amino acids, the building blocks of proteins, and nucleotides, the building blocks of nucleic acids. It is Ammonium nitrate is the preferred form of mineral nitrogen provided to crops in the form of fertilizer. Nitrate-nitrogen is first reduced to nitrite and then to ammonium through the activity of a metabolic pathway common to most herbaceous plants. Depending on the species, part or all of the absorbed nitrate will move to leaf cells through the xylem before it is reduced to ammonium. Ammonium, or other reduced forms of nitrogen are also absorbed (although usually at lower rates) by the root system but their assimilation does not require reduction. These newly absorbed ammonium or ammonium-containing molecules join the endogenous pools in the cells which is formed by ammonium cycling through amino acids and other nitrogenous molecules. Some species do not metabolize nitrate-nitrogen easily and therefore cannot rely on nitrate as sole nitrogen source; many coniferous species fall into this latter category. Legumes and other symbiotic plant species form a third large class of nitrogen user within the plant kingdom; they form a metabolic alliance with a microbial organism through which they can fix gaseous nitrogen. This reduced nitrogen is used efficiently by the plant for growth, and therefore, these crops can develop independently of the availability of mineral nitrogen in the soil.
Many microbes and wild plant species will adapt extensively to availability of nitrogen sources and can therefore complete their life cycle in the absence of one molecular form of nitrogen, which they could use exclusively and efficiently if available in another growing environment. As for most assimilatory pathways, nitrogen assimilation is tightly regulated in cells. As an example, the expression of genes encoding nitrate reductase (NaR) and nitrite reductase (NiR), which are responsible for the reduction of nitrate to ammonium, has been extensively described in various microbial and plant species (for a review, see Miflin and Lea, Books 5 and 12 in The Biochemistry of plants). Although nitrate is not the only regulatory molecule involved in the control of NaR and NiR expression, its presence is essential to initiate the cascade of transduction that eventually leads to sustained transcription and translation of these genes. It has been shown that expression of NaR and NiR genes is repressed in leguminous plants when they are grown in the absence of mineral nitrogen
NiR promoters have been characterized in some plant species (Back et al., 1991, Plant Molecular Biology 17:9-18; Sander et al., 1995, Plant Molecular Biology 27:165-177). Inducibility of these promoters have also been characterized using marker genes in transgenic plants, where it was shown that availability of nitrate is required for full activation of transcription.
Assimilatory pathways for other nitrogen sources have also been described, and promoters for genes involved in some of these pathways have also been characterized.
Genetic transformation of microbes have been used for more than 15 years to produce useful recombinant molecules, and applications in the pharmaceutical, cosmaceutical and dermaceutical industries are being currently exploited. This technology has expanded from microbes to plants and animals in the last ten years with the development of techniques required to adapt this general concept to complex eukaryotic organisms. Basically a gene encoding for a protein of interest or a gene encoding for an enzyme responsible for a modification of a metabolic pathway that leads to a molecule of interest, is linked in an appropriate fashion to cis-and trans-acting regulatory sequences, and transferred to a target cell where it is incorporated in the molecular machinery (in a transitory or stable fashion). The transgenic cell, or a tissue or organism regenerated from the transgenic cell will then perform transcription and translation of the transgene and therefore be enabled to accumulate the protein of interest or to perform the new metabolic reaction through the activity of the enzyme of interest.
The emerging industry of molecular farming (production of recombinant molecules in animals or crops) is one of the most promising industry of the coming century. Its promise is to provide safe and renewable molecule factories for the industry. Among the applications that are currently developed are the production of low-cost monoclonal antibodies for therapeutic and diagnostic uses, the production of unlimited amounts of hormones, cytokines and other bio-active molecules for the treatment of chronicle or lethal diseases, the production of bio-safe substitutes for various blood components, the production of unlimited amounts of processing enzymes for the food and pulp industry, the production of low-cost enzymes for waste treatments, and the production of safe bio-active molecules for the cosmetic industry.
Limitations to the application of this technology has often come from the inability of transgenic organisms to accumulate adequate amounts of the recombinant product, as a result of low transcription rates, improper splicing of the messenger, instability of the foreign mRNA, poor translation rates, hyper-susceptibility of the recombinant protein to the action of endogenous proteases or hyper-susceptibility of the recombinant organism to the foreign protein which result in improper and limited growth or in the worst cases, in strong deleterious effects to the host organism. Inadequacy of production level has a direct impact on the development of applications when profit margins are narrow, or when treatment and/or disposal of residual matter causes bio-safety or environmental problems. Improvement of the accumulation level of the desired recombinant product thus appears to be one critical factor that warrants commercialization of many applications of molecular farming.
The use of inducible promoters has been proposed, and in some instances used successfully, to counteract the combined effect of all the above-mentioned factors. Strong inducible promoters may succeed in generating high ephemerous transcription rates which result in high transitory accumulation of foreign mRNA and translational product. As a result, when inducibility of expression is paired with adequate synchronized protein recovery procedures, the yield per unit obtained is higher than with the use of constitutive expression.
Several expression cassettes harboring inducible promoters have been developed for microbial production systems, and some are currently available for research purposes. Some inducible promoters are currently used in plant (wound inducibility) or animal (specificity to cells of the mammary glands, PPL) systems, although none reported are using low-cost and bio-safe chemical inducers such as nitrate salts.
It would be highly desirable to be provided with a method of regulating the transcription of transgene in genetically-modified organisms.
One aim of the present invention is to provide a method of regulating the transcription of transgene in genetically-modified organisms.
Another aim of the present invention is to provide the use of expression vectors harboring the coding sequence of a gene of interest under the transcriptional control of promoting sequences for which activity is regulated by the presence of nitrogen.
The present invention relates to the use of a nitrogen-inducible expression cassettes for the controlled expression of foreign genes in plants. It will be shown from the following description that isolating such a regulatory sequences can be performed so that when cis-acting sequences are appropriately associated to the open reading frame of a gene of interest, its transcription can be controlled by the addition of specific nitrogen sources.
In one aspect of this invention, the targeted system uses leguminous plant species, so that constructs containing a nitrate-inducible promoter will be maintained transcriptionally low throughout the growth period if the transgenic plant is maintained on a nitrate-free medium, thus allowing the development of the plant biomass without interference from the transgenic trait. Upon addition of nitrate to the growth medium, transcription will be induced in a relatively large proportion of the biomass over the following days. Optimization of induction time and protein accumulation will then be performed in order to maximize recovery of the desired recombinant product.
Although the following description will make clear that this invention can be easily adapted to nitrate induction on nitrate-deprived transgenic leguminous plants, it should be remembered that this general concept can also be applied to the development of other production systems, making profit of the wide variety of nitrogen assimilation systems in the microbial, plant and animal kingdoms.
In one other aspect of this invention, nitrogen inducibility can also be used to maximize protein production in organisms which do not perform nitrogen fixation through symbiotic association, but that can use variable sources of nitrogen (reduced or oxidized) for growth, and thus possess the ability to develop adequately while one of their nitrogen assimilation pathway is inactive due to lack of one nitrogenous substrate in the growing environment. Using an expression cassettes that controls the transcription of any gene in this inactive pathway in order to drive the expression of a gene of interest in such an organism, will allow for inducible expression of the transgenic trait upon addition of the previously lacking nitrogenous compound. As an example, duckweed is a plant species that can grow alternately on nitrate or ammonium; this invention could be used to develop an expression cassette harboring an ammonium-inducible promoter appropriately linked to a gene of interest so that the induction would be performed on nitrate-grown transgenic duckweed plants.
In accordance with the present invention there is provided a method for regulating transcription of a foreign gene in transgenic organisms, comprising the steps of:
a) preparing a transgenic organism using an expression construct consisting of at least a nitrogen-inducible promoter having a sequence selected from the group consisting of SEQ ID NOS: 1 to 13 and functional fragments and derivatives thereof, and an ORF of a gene, wherein said promoter is operationally located with respect to said gene for expression of said gene.
In accordance with a preferred embodiment of the present invention, the method of may further comprise the step of regulating transcriptional expression of said gene by addition or removal of a nitrogen inducer.
In accordance with a preferred embodiment of the present invention, there is provided a method for regulating transcription of a foreign gene in transgenic organisms comprising:
a) preparing an expression construct consisting of at least a nitrogen-inducible promoter with or without cis-acting sequence, an ORF of a gene, and a polyadenylation signal end site at the 3xe2x80x2end of said construct, wherein said promoter is operationally located with respect to said gene for expression of said gene and modulated for transcriptional expression of said gene by addition or removal of a nitrogen inducer;
b) sub-cloning the construct of step a) into a suitable transfection vector for said organism;
c) transferring said vector into DNA of said organism or a cell thereof; and
d) selecting for transgenicity on a suitable medium.
In accordance with a preferred embodiment of the present invention, the method of may further comprise the steps of:
e) introducing the vector into a suitable Agrobacterium tumefaciens strain;
f) using the Agrobacterium strain of step a) to transfer T-DNA into a plant cell;
g) selecting for transgenicity of said plant cell on a suitable medium;
h) regenerating embryos or plantlets from said transgenic cells; and
i) growing mature plants from said regenerated embryos.
In accordance with a preferred embodiment of the present invention, the cis-acting sequence may be isolated from 5xe2x80x2 upstream region of an expressed Nir gene in alfalfa.
In accordance with a preferred embodiment of the method of the present invention, the promoter has the sequence set forth in SEQ ID NO:1 to 13 and functional fragments and derivatives thereof.
In accordance with a preferred embodiment of the present invention, the organism is a plant, more preferably a dicotyledonous plant.
In accordance with a preferred embodiment of the present invention, the organism is alfalfa or tobacco.
In accordance with a preferred embodiment of the present invention, the nitrogen inducer is nitrate.
In accordance with a preferred embodiment of the present invention, the DNA transfer method is any suitable transfer method including DNA bombardment, electroporation, PEG-mediated DNA transfer and whiskers, among others.
In accordance with a preferred embodiment of the present invention, the expression construct comprises at least a nitrogen-inducible promoter and at least one cis- or trans-acting elements.
In accordance with a preferred embodiment of the present invention, the organism is a plant, a fungus, a bacteria, a yeast or an animal.
In accordance with a preferred embodiment of the present invention, the promoter or cis-acting sequence is isolated from the 5xe2x80x2 upstream region of any gene involved in a nitrogen assimilatory pathway.
In accordance with a preferred embodiment of the present invention, the promoter or cis-acting sequence is isolated from the 5xe2x80x2 upstream region of any gene for which transcription is modulated by availability of a given nitrogen source.
In accordance with a preferred embodiment of the present invention, the promoting or cis-acting sequence is any sequence for which transcriptional activity is regulated by addition or removal of any nitrogen source in or from any living organism""s environment.
In accordance with a preferred embodiment of the present invention, the organism from which the promoter or cis-acting sequence is isolated from is any plant, fungus, yeast, bacteria or animal.
In accordance with a preferred embodiment of the present invention, there is provided a promoter for promoting transcription of a foreign gene in transgenic organisms, which comprises a nitrogen-inducible promoter with or without cis-acting sequence for expression of said gene and adapted to be modulated for transcriptional expression of said gene by addition or removal of a nitrogen inducer.
Preferably, the promoter has a sequence selected from the group consisting of SEQ ID NOS: 1 to 13 and functional fragments and derivatives thereof.
In accordance with a preferred embodiment of the present invention, there is provided a terminator allowing expression of a foreign gene in transgenic organisms being used in combination with a promoter, which comprises a polyadenylation signal end site for insertion at the end of said gene, wherein said terminator is operationally located with respect to said gene and said promoter and thereby allows expression of said gene.
Preferably, the terminator has a sequence selected from the group consisting of SEQ ID NOS: 14 to 16 and functional fragments and derivatives thereof.
For the purpose of the present invention the following terms are defined below.
The expression xe2x80x9cfunctional fragments or derivatives thereofxe2x80x9d is intended to mean any derivative or fragment of sequences SEQ. ID. NOS:1-16 which allow for an equivalent level of expression of a foreign gene as the promoter of the present invention set forth in SEQ. ID. NOS:1-13 or as the terminator of the present invention set forth in SEQ. ID. NOS:14-16.